The goal is to determine how hyperthermia affects the types of molecular recombinationaI events and chromosomal aberrations observed when double strand breaks occur either during G1 or during S in replicating or nonreplicating regions of the DNA. At the cytological level, studies will determine the frequencies and types of deletions and exchanges within and between chromosomes that occur in replicating regions vs. nonreplicating regions. In situ hybridization techniques utilizing antibodies to BrdU incorporated into DNA will be utilized. At the molecular level, studies will determine how long range recombinational events involving repeat sequences depend on the particular type of treatment and the cell cycle phase in which the treatment is administered. Emphasis is placed on studying the spectra of mutants when double strand breaks (DSBs) are induced in the HPRT gene at the time the cluster of replicons containing the gene is replicating compared with the spectra when DSBs are induced in the HPRT gene when the cluster of replicons containing the gene has completed replication. For this aim, polymerase chain reaction (PCR) techniques and southern blots will be used to locate deletions in the HPRT gene in the X chromosome. Once the deletions are detected, the breakpoints will be sequenced with PCR primers. These investigations should provide information important for understanding how radiation induces chromosomal aberrations leading to mutations and cell lethality, and how heat enhances radiation-induced chromosomal aberrations, mutations and cell lethality. An understanding of these mechanisms should enable us to improve the design of clinical trials utilizing radiation and hyperthermia.